Plasmacytoid dendritic cells (pDC) or natural interferon type I producing cells play an important role in linking innate and acquired immunity. pDC express high levels of interleukin 3 (IL-3) receptor (CD123) and are present in cord and peripheral blood, T cell areas of the lymph nodes and in the thymus and have the capacity to produce high levels of IFN-a in response to viruses and other stimuli. A depletion of peripheral blood pDC in patients with AIDS and a decrease in IFN-a production with disease progression have been found. Since our preliminary data show that pDC in the thymus are targets for HIV infection and express X4 HIV-1, our general objective is to gain an understanding of the role of pDC in virus spread and immunodeficiency in HIV infection. The present proposal represents a unique collaborative approach to elucidate the effects of HIV infection on pDC development and function, to define the role of pDC in normal T cell development and to determine the mechanisms of interactions of HIV with pDC and thymocytes. We will use our established methods as well as novel approaches and unique reagents to answer the questions presented by the specific aims. Our specific aims are intended to test the hypothesis that HIV infection disturbs the development of pDC and their function thereby affecting normal T cell maturation and immune responses to HIV. The aims are: 1) To investigate the effects of primary HIV isolates on development and function of pDC in the thymus; 2) To define the function of pDC in development of T cells in the thymus; 3) To determine the mechanisms of the interactions of HIV with pDC and developing T cells. The following is an outline of our experimental strategy. We will address how X4 and R5 HIV infection of the thymus affect development of pDC from tagged CD34' cells injected into the HIV-infected SCID-hu mouse and exposure of CD34' cells developing in vitro to HIV or HIV (Env) proteins. Levels of HIV coreceptor expression at different stages of maturation of pDC will be tested by Quantitative FACS analysis. Since pDC produce IFN-a after exposure to HIV we will investigate whether IFN-a influences pDC viability and function and whether IFN-a inhibits human T cell development as observed in the mouse. The role of pDC in negative selection will be evaluated in human and murine fetal thymic organ culture. To determine the mechanism of interaction of HIV on pDC, unique HIV molecular cloned and primary isolates will be used. The binding capacity of HIV virions to thymic pDC as compared to myeloid derived DC will be assessed by novel tetrameric Env constructs. The study of pDC in HIV infection of the thymus provides an opportunity to improve our understanding of HIV induced immunodeficiency.